La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne virus recognized as a major cause of pediatric encephalitis in North America with 70-130 symptomatic cases each year. The virus was first identified as a human pathogen in 1960 after its isolation from a 4 year-old girl who suffered encephalitis and died in La Crosse, Wisconsin. The majority of LACV infections are mild and never reported, however, serologic studies estimate infection rates of 10-30/100,000 in endemic areas. LAC encephalitis has become the most commonly reported pediatric arboviral encephalitis in the US with 70-130 symptomatic cases a year with severe sequelae. For these reasons we are interested in developing a vaccine to prevent this CNS disease. Sequence analysis of the complete LACV genomes of low-passage LACVhuman1960, LACVmosquito1978, and LACVhuman1978 strains and of biologically cloned derivatives of each strain, indicates that circulating LACVs are genetically stable over time and geographic distance with 99.6-100%, 98.9-100%, 97.8-99.6%, and 99.2-99.7% amino acid identity for N, NSs, M polyprotein, and L proteins respectively. [unreadable] Following the intraperitoneal inoculation of mice, LACV replicated in various organs before reaching the CNS where it replicates to high titer causing death from neurological disease. The peripheral site where LACV replicates to highest titer is the nasal cavity, and, presumably, LACV can enter the CNS via the olfactory neurons from nasal olfactory epithelium. The mouse infectious dose50 and lethal dose50 was very similar (<500 PFU) for LACV administered either intranasally or intraperitoneally (moribundity is used as a humane surrogate for lethality in all mouse studies). The ability to efficiently infect mice by the intranasal route raises the possibility that LACV might use this route as one of several ways to infect its natural hosts. It would be very interesting to examine if LACV can be detected in water in tree holes harboring LACV infected mosquito larvae. It is possible that LACV present in the tree hole water (the virus being derived from transovarially infected mosquito larvae) could initiate an infection of the epithelium of the respiratory tract of the mammals that drink the water in the tree holes.[unreadable] LACV was highly infectious for rhesus monkeys and infected 100% of the animals at 10 PFU. However, the infection was asymptomatic, and the level of replication of LACV, as indicated by viremia and titer of virus in lymph nodes, was very low. Despite the low level of identifiable LACV in the monkeys following peripheral inoculation, a strong neutralizing antibody response was elicited. Rhesus monkeys can therefore be used to study the effect of an attenuating mutation on the level of immunogenicity and on the infectivity of a vaccine candidate. In this manner, attenuating mutants that adversely affect immunogenicity or infectivity can be eliminated from further consideration as vaccine candidates. As discussed above, the pathogenesis of LACV for the CNS of rhesus monkeys will be studied following intracerebral inoculation of virus. This will permit the identification of vaccine candidates that are restricted for replication in the CNS of non-human primates, but that are immunogenic and infectious by a peripheral route. [unreadable] A reverse genetics system for LACV has previously been reported by others and was used to generate a virus lacking a functional NSs ORF (NSs KO). We have reproduced this system and have recovered a set of rLACV viruses with the NSs KO mutation, the E577G mutation in GC, or the T148A mutation in GN either as a single mutation or in various combinations. The rLACV mutants are being evaluated for neurovirulence, neuroinvasiveness, and for level of replication in the CNS of mice. Their level of infectivity and immunogenicity will be evaluated in non-human primates to determine if they are infectious, attenuated, and immunogenic for non-human primates. [unreadable] Additional attenuating mutations are being developed for studying the pathogenesis of LACV in mice and for inclusion in a vaccine candidate. Six paired-charge-to-alanine mutations in the N protein have been made, and viruses bearing these mutations have been recovered. These mutants are being further characterized in vitro and in vivo. In addition, numerous LACV mutants have been isolated following growth in the presence of the mutagen 5-FU and have been screened for reduced replication in cells of neural origin. To date, two mutant viruses have been identified that replicate efficiently in Vero cells but are restricted for replication in neural cells and that have reduced lethality for mice in vivo. The genetic basis of attenuation of these mutants is currently being determined, a complicated task since they contain multiple sequence differences in comparison with their wt LACV parent.[unreadable] Jamestown Canyon virus (JCV), family Bunyaviridae, is a mosquito-borne pathogen endemic in the United States and Canada. JCV was first isolated from Culiseta inornata mosquitoes collected near Jamestown Canyon, northwest of Boulder, Colorado. JCV is a member of the California serogroup of viruses in the genus Orthobunyavirus. [unreadable] JCV is an insect-vectored virus with a large geographic range, including much of the United States and Canada. The virus is genetically similar to Inkoo virus circulating in Europe, suggesting most of the northern hemisphere contains JCV or similar variants. The main vectors for the virus are Aedes and Ochlerotatus mosquito species, but the virus has been isolated from 26 species of mosquitoes and 3 species of tabanid flies. In the US, white-tailed deer are the primary amplifying host, but mule deer, sika deer, moose, and bison have also been infected. Antibodies have also been detected in horses and goats. [unreadable] In humans, the virus causes mild febrile illness as well as infection of the CNS resulting in meningitis and encephalitis. Serologic evidence suggests a 4-11% infection rate, with JCV infection more common in some areas than LACV. In humans, JCV disease appears to be milder than LACV and generally affects adults, whereas LACV preferentially causes disease in children. JCV disease is generally associated with headache, fever, neck stiffness, photophobia, nausea, vomiting, and seizures. Although, JCV infection has been diagnosed by PCR of a brain biopsy, reports of human isolates of JCV have not been made. [unreadable] As an initial step in the development of a JCV vaccine, we established both mouse and monkey infection models for wild-type JCV infection. JCV was less neuroinvasive following intraperitoneal inoculation than LACV, however the virus was highly infectious with a resulting mouse infectious dose50 and lethal dose50 of 6 and 60,000 PFU, respectively. The virus was highly neurovirulent with an intracerebral lethal dose50 of 16 PFU.[unreadable] JCV was infectious for rhesus monkeys with 7 of 8 of the animals infected after subcutaneous inoculation with 5 log10 PFU as indicated by a rise in antibody titer. As with LACV, the infection was asymptomatic, however JCV infection did result in a detectable viremia in 6 of 8 monkeys ranging from 5 1000 PFU/mL serum on days 2-4 post-infection. Since both viremia and a neutralizing antibody response are observed, rhesus monkeys can be used to study the effect of an attenuating mutation on the level of replication, immunogenicity, and on the infectivity of a JCV vaccine candidate. In this manner, attenuating mutants that adversely effect immunogenicity or infectivity can be eliminated from further consideration as vaccine candidates. [unreadable] We also determined the antigenic relatedness of LACV and JCV in rhesus monkeys comparing post-infection serum neutralizing antibody titers they are only 3 to 9% related, suggesting that separate vaccines would be needed to optimally protect against LACV and JCV.